General Introduction to Problem Area
Somatic embryogenesis in plants is a process in which somatic embryos are formed from an initial explant being a cell in a plant tissue. The somatic embryos formed are genetically identical copies of the plant providing the initial explant. The process of somatic embryogenesis thereby offers a tool to obtain large numbers of genotypically identical plants for multiplication of selected genotypes of commercial interest, for conservation of endangered species or for generating genetically uniform plant material for research purposes.
Physiological Background to the Procedures Related to the Problem
To produce plants from somatic embryos of conifers, a multi-step procedure is applied to meet the physiological needs of the different stages of development as described below and shown in FIG. 1. Initiation of somatic embryogenesis starts with induction of somatic embryos from an initial explant, typically an immature zygotic embryo, on a solidified culture medium containing plant growth regulator. Somatic embryos continue to form, typically on the same composition culture medium, and a proliferating embryogenic culture form. At the proliferating stage, several of the key features generally regarded as beneficial for the process of somatic embryogenesis process, take place: (i) the mass propagation of genotypically identical propagules through unlimited multiplication of immature somatic embryos; (ii) cryogenic storage of proliferating embryos substantiates an virtually eternal store of clones, i.e. a clone bank is established, (iii) transgenic modification of the immature somatic embryo allow for large scale propagation of genetically improved propagules. At the next step in the procedure, the proliferating somatic embryo is subjected to a growth medium that triggers embryo development to progress into the maturation stage. Conversion from proliferation to maturation only occurs in a fraction of the proliferating embryos in the culture. Low conversion rates are encountered more frequently in genotypes from recalcitrant conifer species, but are common in all conifer species as well as other plant species. The manual labour needed to collect embryos increase with the decrease in conversion rate, and thereby the cost and risk of contamination and other inaccuracies. Low conversion rate from proliferation to maturation is a major bottleneck for commercial large scale applications of somatic embryogenesis procedures. For germination, mature somatic embryos are subjected to different culture regimes to induce root- and shoot formation, in a number of different steps; desiccation, sucrose treatment, red light induction, and blue light stimulation. Thereafter, germinated embryos deemed appropriately developed are transferred to a compost material and gradually transferred to an environment ex vitro during which the sucrose content is reduced. The different treatments during germination into a plant requires repeated manual handling of individual germinants and plants adding a considerable cost to the overall procedure.
Production of Plants from Somatic Embryos
The prior art procedure for producing plants from somatic embryos requires manual handling at several steps making the procedure time consuming, expensive and inaccurate.
For conifer species, standard procedures used involve several steps when manual handling is required. The general procedure is outlined in FIG. 1 (see e.g. von Arnold S, Clapham D. Spruce embryogenesis. 2008. Methods Mol Biol. 2008; 427:31-47; Belmonte M F, Donald G, Reid D M, Yeung E C and Stasolla C. 2005. Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca). J Exp Bot, Vol. 56, No. 419, pp. 2355-2364).
There are four steps that rely on manual handling to obtain a small plant from the mature somatic embryo as seen in FIG. 1. The first manual interaction is when [1] the mature embryo is isolated from immature embryos (120), and placed horizontally in a plastic container under sterile conditions; the second [2] occur after 3-7 days of resting (130), then mature embryo is transferred to a gelled culture medium for initiation of germination processes. The germinated somatic embryo will under appropriate culture medium composition and light conditions initiate roots (140). The third manual transfer [3] is when the germinant having a small root formed is transferred to an upright position with the root partially immersed in liquid germination media (150). The fourth [4] and final transfer is when the germinated embryos has a tap root and small lateral roots, then it is transferred into a solid substrate in a pot for further plant formation (160).
TABLE 1List of designations pertaining to FIG. 1.ItemDesignation100Mature embryo101Crown of a mature embryo102Foot of a mature embryo103Width of crown of a mature embryo104Length of a mature embryo120Maturation phase130Resting phase140Germination phase150In vitro plant formation phase160Ex vitro plant formation phase
Conversion from proliferation to maturation only occurs in a fraction of the proliferating embryos in the culture. Low conversion rates are encountered more frequently in genotypes from recalcitrant conifer species, but are common in all conifer species as well as other plant species. The manual labour needed to collect embryos increase with the decrease in conversion rate, and thereby the cost and risk of contamination and other inaccuracies. Low conversion rate from proliferation to maturation is a major bottleneck for commercial large scale applications of somatic embryogenesis procedures. For germination, mature somatic embryos are subjected to different culture regimes to induce root- and shoot formation, in a number of different steps; desiccation, sucrose treatment, red light induction, and blue light stimulation. Thereafter, germinated embryos deemed appropriately developed are transferred to a compost material and gradually transferred to an environment ex vitro during which the sucrose content is reduced. The different treatments during germination into a plant requires repeated manual handling of individual germinants and plants adding a considerable cost to the overall procedure.
Plant embryos can be suspended into a liquid to facilitate automated processing. However, the liquid-suspended embryos become randomly oriented and need to be oriented properly at planting.
U.S. Pat. No. 5,284,765A discloses a method of orienting plant embryos. The properly desiccated embryos are suspended in a benign liquid flotation medium having a density in the range of about 1.059-1.104 g/cm3. The density must be adjusted empirically so that a predominant number of viable embryos will float and nonviable embryos will sink. In at least the case of conifer somatic embryos, they will float with the end bearing the latent cotyledons upward. After sufficient separation time in the flotation medium the oriented embryos are swept by a flowing liquid stream into a conduit. They enter cotyledon end first and are then carried to a delivery point without losing that orientation. Here they are separated from the transporting medium. The embryos, still positioned cotyledon end first, may then be picked up by robotic or other means for further processing, such as insertion into an artificial seed. However, the method is laborious e.g. in that the density of the flotation medium needs to be experimentally determined. Also, the sorting of viable and nonviable embryos is quite inexact.
US2002192040A discloses apparatus and methods useful for introducing a desired spacing between or classifying and sorting objects, e.g. plant embryos. Objects carried serially in a fluid stream enter the apparatus via an upstream conduit. A sensor associated with the conduit provides information regarding an object at a particular location in the upstream conduit and produces a signal. A switch coupled to the upstream conduit directs the fluid stream to a downstream conduit designated for a certain type of object in response to the signal by applying a force to a conduit, e.g., by aligning the upstream conduit with a downstream conduit to create a fluid-tight path. However, the apparatus does not provide means of orientating the embryos.
It would be desirable to obtain a means for efficient and accurate orientation of embryos. The present invention relates to an apparatus which allows automatization of the step of orienting the embryos correctly prior to planting. It is an object of the invention to provide an automated apparatus for orienting plant embryos.